R01HL103827-06A1 Resubmission (Zhou, Lan) 10.22.2018 Abstract Hematopoietic stem cell (HSC) transplantation is the only curative option for various malignant and a few nonmalignant diseases. A successful outcome is dependent on infusing an adequate number of functionally active mobilized hematopoietic progenitor cells (HPCs). Inadequate mobilization in patients showing poor responses to current mobilizing agents remains a clinical problem. In addition, transplantation using lower-then-desired doses of stem cells increases risks of stem cell engraftment failure. Hence, developing more efficacious and low-risk HPC mobilization regimens and strategies will greatly improve patient outcomes. We recently identified a novel role of Notch as a cell surface adhesion as well as signaling molecule to retain stem cell in the bone marrow microenvironment. Notch is a conserved cell surface receptor that regulates stemness, growth, and differentiation. Notch transactivation is the result of functional engagement of Notch receptors with Notch ligands, which is dependent on the posttranslational modification of Notch receptors with O-fucose and Fringe on the epidermal growth factor (EGF) modules of Notch extracellular domain. Structural studies have demonstrated that O-fucose attached to the specific threonine residue of the core ligand binding region of Notch EGF repeats functions as a surrogate amino acid to make specific and functional contact to Notch ligand DLL4 and JAG1, respectively. Elongation of O-fucose to a disaccharide (GlcNAc?1- 3Fucose) by Fringe further increases Notch binding affinity to some Notch ligands. In the last funding cycle, we identified conservative Notch-ligand adhesive interaction and its modification by O- fucosylation for HSC quiescence and niche maintenance. We provided compelling evidences showing that deficiency of O-fucosylation leads to decreased HSC quiescence and HSC adhesion to bone marrow niche cells, and increased HSC egress from the marrow. We also reported that neutralizing DLL4 or JAG1 as well as blocking Notch2 significantly enhances HSC egress to the periphery. More, recently, we found transient Notch2 blockade results in superior engraftment and hematopoietic recovery of mobilized HSPC associated with enhanced HSPC activity and attenuated ER stress activation. Further, we found that Fringe-modified recombinant Notch peptides bearing the core ligand binding EGF repeats function as decoys for Notch ligand to induce HSC and progenitor cell egress in osteoblastic spheroids. We thus hypothesize that Notch2 interaction with DLL4 or JAG1 regulates ligand- and niche-specific HSPC retention. We further hypothesize that Notch2-targeted mobilization regimen may improve HCT outcome by mitigating ER stress response activation. We will test this hypothesis in three aims. In Aim 1, we will examine the differential roles of JAG1 and DLL4 expressed by the endothelial and the immature osteolineage cells in the regulation of HSC niche retention, location and mobilization. In Aim 2, we will assess the ligand binding affinity and the efficiency of Fringe-modified Notch peptides as decoys to enhance HSPC egress from HSC niche. In Aim 3, we will examine the significance and the mechanism by which blocking Notch2 potentiates HSPC activity through attenuating ER stress response. At the end of this study, we hope our findings will reveal novel aspects of Notch signaling in HSC niche biology and in ER stress regulation which may provide a resource for understanding Notch signaling in the control of HSC activity during stress hematopoiesis, with implications for identifying ways to improve HCT outcomes.